Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in this process. In particular we are studying the ubiquitous molecular chaperone Hsc70 and the J-domain cofactor proteins that induce specific substrates to bind to Hsc70. In our previous work we have studied the role of Hsc70 in clathrin-mediated endocytosis, in particular its ability to dissociate clathrin from clathrin-coated vesicles. We first discovered that uncoating not only requires Hsc70 but also the 100 kDa nerve-specific J-domain protein auxilin or the non-neuronal homolog of auxilin, the 150 kDa protein GAK that is similar to auxilin but also contains an N-terminal kinase domain. We then showed that in vivo clathrin-coated pits are dynamic structures and both clathrin and other components of clathrin-coated pits including the clathrin adaptor protein AP2 exchange during clathrin-mediated endocytosis. Similarly, clathrin and the clathrin adaptor protein AP1 on the trans-Golgi network exchanges with free clathrin and AP1 in the cytosol. From our data we concluded that clathrin exchange is required for the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. We then showed using permeabilized cells that Hsc70 not only dissociates clathrin after clathrin-coated vesicles bud off but is also required for the clathrin exchange that occurs during invagination of clathrin-coated pits on the plasma membrane or clathrin-coated buds on the TGN. [unreadable] [unreadable] During the past year our laboratory has characterized mouse embryonic fibroblasts derived from GAK K/O mice. When we examined transferrin uptake in the cre-treated MEFs we found that transferrin uptake was almost completely blocked. Consistent with these results, the transferrin receptor had a diffusive appearance over the plasma membrane with no internalized pool of transferrin visible. When we then examined the status of the CCPs in the cell, we found that there were very few clathrin puncta on the plasma membrane, while the AP-2 had an aberrant distribution, appearing clustered, along with epsin, eps15 and dynamin. When the MEFs were transfected with GFP-clathrin, the few remaining clathrin puncta on the plasma membrane showed no clathrin exchange. Therefore, these results show that deletion of GAK causes a profound disruption in clathrin-mediated endocytosis, which is most likely the cause of the lethality that we observe in developing and mature mice in which GAK has been knocked out in specific tissues. [unreadable] [unreadable] We have also carried out studies on primary neurons cultured from auxilin knock-out mice. Western blots showed that these neurons had a marked increase in clathrin and in agreement with these results, light microscopy showed an increase in clathrin structures in the cell. More specifically, electron microscopy studies showed a marked increase in clathrin-coated vesicles in the cells. These results are consistent with auxilin being required for rapid uncoating of clathrin-coated vesicles in the neurons; apparently even in the presence of GAK, auxilin is required for this process. Apparently, in the absence of auxilin, the cell compensates for the clathrin trapped in clathrin-coated vesicles by increasing the total amount of clathrin in the cell. [unreadable] [unreadable] In a related study, on the mechanism of formation of clathrin-coated pits in cells, we have investigated the interaction of clathrin with GGAs, a class of monomeric clathrin adaptors involved in the sorting of cargo at the trans-Golgi network of eukaryotic cells. Previous studies have shown that GGAs interact with clathrin both in solution and in the cell, but it has not yet been shown whether they assemble clathrin. We find that GGA1 promoted assembly of clathrin with complete assembly achieved when one GGA1 molecule is bound per heavy chain. In the presence of excess GGA1, we obtained the unusual stoichiometry of five GGA1s per heavy chain, and even at this stoichiometry the binding was not saturated. The assembled structures were mostly baskets, but approximately 10% of the structures were tubular with an average length of 180 +/- 40 nm and width of approximately 50 nm. From these results we conclude that the clathrin adaptor GGA1 is a clathrin assembly protein unique in its ability to polymerize clathrin into tubules.[unreadable] Finally, we have studied the effect of normal cellular prion protein (PrP(C)) on abnormal protein aggregation by transfecting huntingtin fragments (Htt) into SN56 neuronal-derived cells depleted of PrP(C) by RNA interference. PrP(C) depletion caused an increase in both the number of cells containing granules and the number of apoptotic cells. Consistent with the increase in Htt aggregation, PrP(C) depletion caused an decrease in proteasome activity and a decrease in the activities of cellular defense enzymes compared with control cells whereas reactive oxygen species (ROS) increased more than threefold. Therefore, PrP(C) may protect against Htt toxicity in neuronal cells by increasing cellular defense proteins, decreasing ROS and increasing proteasome activity thereby increasing Htt degradation. Depletion of endogenous PrP(C) in non-neuronal Caco-2 and HT-29 cells did not affect ROS levels or proteasome activity suggesting that only in neuronal cells does PrP(C) confer protection against Htt toxicity. The protective effect of PrP(C) was further evident in that overexpression of mouse PrP(C) in SN56 cells transfected with Htt caused a decrease in both the number of cells with Htt granules and the number of apoptotic cells, whereas there was no effect of PrP(C) expression in non-neuronal NIH3T3 or CHO cells. Finally, in chronically scrapie (PrP(Sc))-infected cells, ROS increased more than twofold while proteasome activity was decreased compared to control cells. Although this could be a direct effect of PrP(Sc), it is also possible that, since PrP(C) specifically prevents pathological protein aggregation in neuronal cells, partial loss of PrP(C) itself increases PrP(Sc) aggregation.